The goal of this research project is to examine the biochemical properties and biological effects of the major beta-amyloid (Abeta) isoforms in cell culture models and to explore potential mechanisms of amyloid deposition in Alzheimer's disease and normal aging. Recent work suggests that the longer major Abeta isoform, Abeta1-42, may be more intimately associated with AD pathology than the shorter form, Abeta1-40. Our investigations of the biochemical properties of these peptides has established that Abeta1-42 is significantly less soluble than Abeta1-40 and shorter isoforms. Unexpectedly, we also discovered a major biological difference in the capacity of cells to catabolize Abeta1-42 compared to Abeta1-40 and the shorter Abeta- peptides. In the previous award period, we have extended these observations to the interaction of Abeta with differentiated PC12 cells. We found that Abeta1-42 is internalized by PC12 cells in vitro and accumulates intracellularly in late endosomes or lysosomes. Although the shorter Abeta analogs are also internalized by PC12 cells, they are degraded or eliminated and do not accumulate. This specific accumulation of Abeta1-42 is largely due to the resistance of the internalized Abeta to degradation. Unlike our results with human fibroblasts, we found that a significantly larger amount of Abeta1-42 relative to Abeta1-40 is adsorbed to the surface of PC12 cells. We have also examined the effect of Abeta1-42 on the processing and catabolism of APP. The results of these investigations demonstrate that Abeta1-42 dramatically stimulates the accumulation of amyloidogenic fragments of APP, particularly a 16 kDa fragment, in an insoluble fraction of the cell. The specific aims of this proposal are designed to compare the biological properties of Abeta1-42 and Abeta1-40 and more completely characterize the effects of Abeta1-42 on APP catabolism and Abeta secretion and to test whether these effects may be relevant to the accumulation of insoluble amyloid deposits in AD. We propose to develop a facile and quantitative assay to distinguish Abeta1-42 and Abeta1-40 in biological samples which takes advantage of the unique biochemical properties of the longer isoform. We will characterize differences in the adsorption, internalization and catabolism of Abeta1-42 and Abeta1-40 in cultured PC12 cells. We will extend our preliminary observations on the effects of Abeta1-42 on the processing and catabolism of APP in transfected 293 cells to cultured PC12 cells. We will also determine whether there is a precursor-product relationship between the 16 kDa amyloidogenic APP fragment and Abeta in the insoluble fraction of cells containing intracellular Abeta1-42 aggregates. We will determine whether the 4 kDa Abeta product is Abeta1-42. We will determine whether the internalization and accumulation of Abeta1-42 impairs endocytic trafficking and normal lysosomal function.